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J. Biol. Chem., Vol. 276, Issue 38, 35883-35890, September 21, 2001

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Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G₁₁ following Fusion to the _1b-Adrenoreceptor

PALMITOYLATION OF G₁₁ IS NOT REQUIRED FOR INTERACTION WITH · COMPLEX^*

Patricia A. Stevens, John Pediani, Juan J. Carrillo, and Graeme Milligan^§

From the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

Received for publication, April 27, 2001, and in revised form, June 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transfection of either the _1b-adrenoreceptor or G₁₁ into a fibroblast cell line derived from a G_q/G₁₁ double knockout mouse failed to produce elevation of intracellular [Ca²⁺] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the _1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S G₁₁ also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G₁₁ or C10S G₁₁. Expression of a fusion protein between the _1b-adrenoreceptor and G₁₁ allowed [Ca²⁺]_i elevation, and this was also true for a fusion protein between the _1b-adrenoreceptor and C9S,C10S G₁₁, since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin , as a ·-sequestering agent, fully attenuated the Ca²⁺ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G₁₁ were also targets for agonist-regulated [³H]palmitoylation and bound [³⁵S]guanosine 5'-3-O-(thio)triphosphate (GTPS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [³⁵S]GTPS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release · complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca²⁺ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein · complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The phosphoinositidase C-linked G proteins G_q and G₁₁ are widely co-expressed (1-6). To gain insight into their function and into potential functions of G protein-coupled receptors (GPCRs)¹ in the absence of these G proteins, their genes have been inactivated in mice (6-12). Although double G_q/G₁₁ knockout mice are not viable, cells derived from such embryos have been extremely useful, particularly in the elucidation of function of the G₁₂/G₁₃ class of ubiquitously expressed, pertussis-insensitive G proteins (11, 12). Such cells have also been used to demonstrate that agonist-induced internalization of G_q/G₁₁-coupled GPCRs is not dependent upon the presence of these G proteins (13).

Post-translational palmitoylation close to the N terminus of the  subunits of heterotrimeric G proteins appears to be central to their effective interaction with the plasma membrane and thus their capacity to transduce signals from GPCRs to effectors (14-16). Since the thioester bond between the protein and the fatty acid is easily cleaved, there is the potential for dynamic regulation of G protein acylation. This has been best examined for the adenylyl cyclase stimulatory G protein G_s, where activation mediated by GPCRs or cholera toxin has been shown to alter [³H]palmitoylation of the G protein (17-19). G₁₁ possesses two adjacent cysteine residues at positions 9 and 10, which have been established to be sites of acylation (20, 21). Mutation of these two cysteine residues results in production of a soluble polypeptide. Few reports have provided evidence for regulation of the palmitoylation of G_q/G₁₁, although elevated incorporation of [³H]palmitate into these G proteins by stimulation of the gonadotrophin-releasing hormone receptor has been reported (22) and by molecularly undefined receptors for 5-hydroxytryptamine in rat brain cortical membranes (23).

Production of fusion proteins between GPCRs and G protein  subunits has become a popular means to explore many aspects of the detailed interactions between these protein classes (24, 25). Because the N terminus of the G protein  subunit is fused directly to the C terminus of the GPCR in such constructs, it is often unclear whether this might limit interaction with the G protein · complex. This uncertainty reflects that the N terminus of the  subunit is an important contact interface for ·, although key amino acids for this interaction are thought to be located some 15 amino acids away from the site(s) of palmitoylation (26).

Herein, we use fusion proteins between the _1b-adrenoreceptor and forms of G₁₁ to demonstrate that the fusion proteins are activated and regulate their palmitoylation status in response to agonist. By examining fusion constructs in which either the receptor or the G protein is resistant to palmitoylation, we also demonstrate that the acylation status of both polypeptide partners is dynamically regulated by agonist. Moreover, palmitoylation of G₁₁ is not necessary for the binding and release of · complex and further transduction of the signal.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A fibroblast cell line (EF88) derived from a combined G_q/G₁₁ double knockout mouse (9-10, 13) was the gift of Dr. M. I. Simon (California Institute of Technology, Pasadena, CA).

[9,10-³H]Palmitic acid was obtained from Amersham Pharmacia Biotech. [³H]Prazosin and [³⁵S]GTPS were purchased from PerkinElmer Life Sciences. Dulbecco's modified Eagle's medium (DMEM), newborn calf serum, L-glutamine, and trypsin/EDTA were purchased from Life Technologies, Inc. Fura-2/AM, phenylephrine HCl, phentolamine, HEPES, Bordetella pertussis toxin, and EGTA were purchased from Sigma. CQ (C terminus of G_q) antisera have previously been described (27, 28).

Construction of Fusion Proteins-- Production and subcloning of wild type and palmitoylation-resistant _1b-adrenoreceptor-G₁₁ fusion proteins was performed in two separate stages. In the first step, the coding sequence of each form of G₁₁ (20, 21) was modified by PCR amplification using the amino-terminal primer 5'-GAGGACGGTACCACTCTGGAGTCCATG-3' the initiating Met of G₁₁ was removed, and both a KpnI restriction site (underlined) and a two-amino acid spacer (Gly-Asn) were introduced. Using the C-terminal primer 5'-TTGTGCGGCCGCCGGTCACACCAGGTT-3, a NotI restriction site (underlined) was introduced downstream of the stop codon of G₁₁. The amplified fragments digested with KpnI and NotI were subcloned into similarly digested pcDNA3 expression vector (Invitrogen). To obtain the various _1b-adrenoreceptor-G₁₁ fusion proteins, the coding sequence of the wild type or C365A, C367G hamster _1b-adrenoreceptor was amplified by PCR. Using the amino-terminal primer 5'-GACGGTACCTCTAAAATGAATCCCGAT-3', a KpnI restriction site (underlined) was introduced upstream of the initiator Met. Using the carboxyl-terminal primer 5'-GTCCCTGGTACCAAAGTGCCCGGGTG-3', a second KpnI restriction site (underlined) was introduced immediately upstream of the stop codon. Finally, the G₁₁ constructs in pcDNA3 were digested with KpnI and ligated together with the PCR product of the _1b-adrenoreceptor amplification also digested with KpnI. The open reading frames thus produced represent the coding sequence of either _1b-adrenoreceptor-G₁₁, C365A,C367G _1b-adrenoreceptor-G₁₁, or _1b-adrenoreceptor-C9S,C10S G₁₁. Each was fully sequenced before its expression and analysis.

Transient Transfection of HEK293 Cells-- HEK293 cells were maintained in DMEM supplemented with 0.292 g/liter L-glutamine and 10% (v/v) newborn calf serum at 37 °C in a 5% CO₂ humidified atmosphere. Cells were grown to 60-80% confluence before transient transfection in 60-mm dishes. Transfection was performed using LipofectAMINE reagent (Life Technologies) according to the manufacturer's instructions.

³H Palmitoylation-- Cells were labeled with 0.5 mCi/ml [9,10-³H]palmitic acid in DMEM supplemented with 0.292 g/liter L-glutamine, 5% (v/v) dialyzed newborn calf serum, and 5 mM pyruvic acid at 37 °C in a 5% CO₂ humidified atmosphere. After incubation for the appropriate time in the presence and absence of varying concentrations of phenylephrine, reactions were terminated by the addition of 200 µl of 1% (w/v) SDS. Proteins were denatured by passage through a 25-gauge needle followed by 5-min incubation at 100 °C. After chilling to 4 °C, 800 µl of Kahn solubilization buffer (1% (v/v) Triton X-100, 10 mM EDTA, 100 mM NaH₂PO₄, 10 mM NaF, 50 mM HEPES (pH 7.2)) was added, and the samples were precleared by incubation for 1 h at 4 °C with 100 µl of Pansorbin (Calbiochem). The precleared supernatants were then incubated for 16 h at 4 °C with protein-A-Sepharose and 10 µl of antiserum CQ (27, 28). Immune complexes were isolated by centrifugation, washed three times with Kahn immunoprecipitation buffer (1% (v/v) Triton X-100, 100 mM NaCl, 100 mM NaF, 50 mM NaH₂PO₄, 50 mM HEPES (pH 7.2) plus 0.5% SDS), and eluted from the protein A-Sepharose by the addition of electrophoresis buffer containing 20 mM dithiothreitol and heating to 80 °C for 3 min. Analysis was by SDS-polyacrylamide gel electrophoresis, using 10% (w/v) polyacrylamide resolving gels and by autoradiography.

[³⁵S]GTPS Binding-- [³⁵S]GTPS binding experiments were initiated by the addition of 10 µg of membranes to an assay buffer (20 mM HEPES (pH 7.4), 3 mM MgCl₂, 100 mM NaCl, 1 µM guanosine 5'-diphosphate, 0.2 mM ascorbic acid, 50 nCi of [³⁵S]GTPS) containing the indicated concentrations of phenylephrine. Nonspecific binding was determined in the same conditions but in the presence of 100 µM GTPS. Reactions were incubated for 15 min at 30 °C and were terminated by the addition of 0.5 ml of ice-cold buffer, containing 20 mM HEPES (pH 7.4), 3 mM MgCl₂ and 100 mM NaCl. The samples were centrifuged at 16,000 × g for 15 min at 4 °C, and the resulting pellets were resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1 mM EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecylsulfate. Samples were precleared with Pansorbin (Calbiochem), followed by immunoprecipitation with CQ antiserum. Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [³⁵S]GTPS was estimated by liquid-scintillation spectrometry.

[³H]Prazosin Binding Studies-- Binding assays were initiated by the addition of 3 µg of cell membranes to an assay buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM MgCl₂, pH 7.4) containing [³H] prazosin (0.05-10 nM in saturation assays and 0.5 nM for competition assays) in the absence or presence of increasing concentrations of phenylephrine (200-µl final volume). Nonspecific binding was determined in the presence of 100 µM phentolamine. Reactions were incubated for 30 min at 30 °C, and bound ligand was separated from free by vacuum filtration through GF/B filters. The filters were washed twice with assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

[Ca²⁺] Imaging-- A fibroblast cell line, (EF88), derived from the embryos of mice in which the  subunits of both G_q and G₁₁ had been knocked out by targeted gene disruption (9-10, 13) were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and L-glutamine (1 mM) in a 95% air and 5% CO₂ atmosphere at 37 °C. For transfection experiments, a portion of the cells harvested during trypsinization were plated onto glass coverslips (22-mm diameter, grade 0 thickness), and after a 24-h growth period, cells were transfected using LipofectAMINE (Life Technologies) according to the manufacturer's instructions. After 3 h, cells were washed twice with OPTI-MEM-1 and then cultured in DMEM growth medium for a further 24 h. In some experiments, after an initial 24-h transfection/growth period, the transfected cells were treated with pertussis toxin (25 ng/ml, 24 h).

Measurement of [Ca²⁺]_i-- Transfected cells growing on coverslips were loaded with the Ca²⁺-sensitive dye Fura-2 by incubation (15-20 min, 37 °C) in physiological control saline solution, 130 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, 10 mM D-glucose, pH adjusted to 7.4 using NaOH) containing the dye's membrane-permeant acetoxymethylester form (1.0 µM). A rise in [Ca²⁺]_i causes a corresponding rise in the Fura-2 fluorescence ratio recorded from cells loaded with this dye, which allows receptor-mediated changes in [Ca²⁺]_i to be monitored using standard, microspectrofluorimetric techniques (29). An Optoscan monochromator (Cairn Research, Faversham, Kent, UK) was used to alternate the excitation wavelength between 340 and 380 nm (band pass of 10 nm) and to control the excitation frequency. Fura-2 fluorescence emission at 510 nm was monitored either by a low noise COHU CCD camera or a photomultiplier tube with a bialkali photocathode. Images acquired with the CCD camera were stored and analyzed digitally under the control of Meta Fluor imaging software (Universal Imaging Corp., West Chester, PA).

Agonist-evoked [Ca²⁺]_i responses were quantified by peak height (i.e. difference between the base-line resting ratio level and that attained at the peak response). Responses were pooled and are expressed as the mean ± S.E. of at least five experiments, with vertical lines (see Fig. 3) representing S.E. Statistical significance of any difference between means was determined using Student's t test. Differences in the magnitude of [Ca²⁺]_i responses evoked by phenylephrine in untreated and pertussis toxin-treated cells were evaluated by Student's paired t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells of a fibroblast-derived line (EF88) from the embryo of a combined G_q/G₁₁ double knockout mouse were grown on glass coverslips. These were transiently transfected with either the hamster _1b-adrenoreceptor or the mouse G protein G₁₁. Co-transfection with enhanced green fluorescent protein allowed identification of positively transfected cells. Following loading of the cells with the Ca²⁺ indicator Fura-2/AM (1 µM, 15 min, 37 °C), single cell Ca²⁺ imaging was performed in the absence of external Ca²⁺. In both cases, the addition of the ₁-adrenoreceptor-selective agonist phenylephrine (10 µM) failed to alter basal intracellular [Ca²⁺] ([Ca²⁺]_i) (Fig. 1, A and B). However, co-expression of both the _1b-adrenoreceptor and G₁₁ resulted in a robust and rapid elevation of [Ca²⁺]i (Fig. 1, C and D). We have previously demonstrated that G₁₁ can be post-translationally acylated on both Cys⁹ and Cys¹⁰ (20) and that mutation of both of these amino acids to Ser prevents membrane association of the G protein (20, 21). Co-transfection of EF88 cells with the _1b-adrenoreceptor and C9S,C10S G₁₁ thus also failed to result in a phenylephrine-mediated elevation of [Ca²⁺]_i (Fig. 2). Equivalent studies with either C9S G₁₁ or C10S G₁₁ did produce an agonist-dependent rise in [Ca²⁺]_i (Fig. 2), but the magnitude of the response was substantially less than with wild type G₁₁ and was kinetically much slower. In both of these regards, C10S G₁₁ performed more poorly than C9S G₁₁ (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Co-expression of the 1b-adrenoreceptor and the G protein G11 is required to elevate [Ca2+] levels in EF88 cells. EF88 cells were transiently transfected with either the hamster 1b-adrenoreceptor (A) or the mouse G protein G11 (B) or co-transfected with the 1b-adrenoreceptor and G11 (C). Green fluorescent protein was co-expressed as a marker for positively transfected cells. Cells were loaded with Fura-2/AM and [Ca2+]i levels imaged before and during exposure of the cells to phenylephrine (Phe; 10 µM). Representative images of basal and peak [Ca2+]i are displayed for two cells co-expressing 1b-adrenoreceptor and G11 (D). Warmer colors represent higher [Ca2+].

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Palmitoylation potential of G11 determines functional interactions with a co-expressed 1b-adrenoreceptor. EF88 cells were transiently transfected with the hamster 1b-adrenoreceptor and each of the following: wild type G11 (1), C9S G11 (2), C10S G11 (3), and C9S,C10S G11 (4). Positively transfected cells were identified by co-expression of green fluorescent protein. The capacity of phenylephrine (3 µM) to elevate [Ca2+]i levels was then imaged. Data are the traces from six individual cells for each set of transfections.

To potentially overcome these deficits, fusion proteins were constructed between the _1b-adrenoreceptor and forms of G₁₁. The G protein sequence was attached directly to the C-terminal tail of the receptor cDNA from which the stop codon was eliminated. This allows production of single open reading frames containing the features of both polypeptides (24, 25). Expression in EF88 cells of the chimeric polypeptide containing the wild type sequences of both receptor and G protein resulted in an elevation of [Ca²⁺]_i upon the addition of phenylephrine (Fig. 3A), although the kinetics of the response were markedly slower than for the isolated but co-transfected receptor and G protein. Now the same was true when a fusion protein between the _1b-adrenoreceptor and C9S,C10S G₁₁ was used (Fig. 3B). This was also the case when a C365A,C367G _1b-adrenoreceptor-G₁₁ fusion protein was expressed (data not shown). Elevation of [Ca²⁺]_i in response to activation of a GPCR can proceed from activation of members of the phosphoinositidase C family by either  subunits of the G_q/G₁₁ family or · complexes (Fig. 4A). To ascertain if the signal from the fusion proteins derived from the receptor-attached G protein  subunit, EF88 cells were co-transfected with the _1b-adrenoreceptor-G₁₁ fusion protein and transducin . Transducin  is used regularly as a ·-sequestering agent, and in this situation the effect of phenylephrine was fully attenuated (Fig. 3A). The N-terminal region of G subunits is an important binding interface for · (30-31). However, transducin  also fully attenuated the phenylephrine signal from the _1b-adrenoreceptor-C9S,C10S G₁₁ fusion protein (Fig. 3B).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Fusion proteins between the 1b-adrenoreceptor and both wild type G11 and C9S,C10S G11 are functional. Elevation of [Ca2+]i is produced by the · complex. Fusion proteins between the 1b-adrenoreceptor and either wild type G11 (n = 10) (A) or C9S,C10S G11 (n = 6) (B) were expressed in EF88 cells. The fusion proteins were co-expressed without (1) or with (2) transducin  (wild type G11, n = 11; C9S,C10S G11, n = 16). The capacity of phenylephrine (3 µM) to modulate [Ca2+ ]i levels was measured as in Figs. 1 and 2. In Fig. 3A, the peak [Ca2+]i induced by phenylephrine in the presence (+) or absence () of transducin  is also displayed for representative cells. Warmer colors represent higher [Ca2+].

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Elevation of [Ca2+]i by the 1b-adrenoreceptor-G11 fusion protein is not via activation of Gi family G proteins. A, receptor-mediated elevation of [Ca2+]i may proceed via either G protein  subunits or the · complex. The ·-mediated elevation of [Ca2+]i (Fig. 3) could potentially derive from release of · from the 1b-adrenoreceptor-G11 fusion protein or from pertussis toxin (Ptx)-sensitive, Gi family G proteins expressed endogenously by EF88 cells. B, membranes from either EF88 (2) or HEK 293 (1) cells were immunoblotted to detect the presence of G11/Gq (upper panel) or Gi (lower panel). C, EF88 cells expressing the 1b-adrenoreceptor-G11 fusion protein were treated with pertussis toxin (25 ng/ml) or with vehicle for 24 h. The capacity of phenylephrine to elevate [Ca2+]i was then measured.

Elevation of [Ca²⁺]_i could also potentially arise from · complex released by interaction of the fusion proteins with members of the pertussis toxin-sensitive G_i family (Fig. 4A), which, unlike G_q and G₁₁, are expressed in EF88 cells (Fig. 4B). Expression of receptors in heterologous systems can result in a reduction in specificity of G protein coupling (32). To eliminate this possibility, experiments were repeated following sustained treatment with pertussis toxin of EF88 cells that had been transfected to express the _1b-adrenoreceptor-G₁₁ fusion protein. This did not alter the phenylephrine-mediated elevation of [Ca²⁺]_i (Fig. 4C). The combined data of Figs. 3 and 4 demonstrate that the _1b-adrenoreceptor-G₁₁ fusion protein both binds endogenous · and is able to release it upon agonist occupancy and that the palmitoylation status and potential of G₁₁ does not limit either its binding or release of · complex.

To directly explore palmitoylation of the fusion proteins and its regulation, _1b-adrenoreceptor-G₁₁ was expressed transiently in HEK293 cells. Both these and mock-transfected cells were labeled with [³H]palmitate for 2 h, and the samples were immunoprecipitated with an antiserum (CQ) that identifies the C-terminal 10 amino acids shared by G_q and G₁₁ (27, 28). Following SDS-polyacrylamide gel electrophoresis and autoradiography, a band of some 42 kDa was observed in both mock-transfected and positively transfected cells (Fig. 5). This corresponds to a mixture of G_q and G₁₁, which are co-expressed by HEK293 cells and not resolved by the gel conditions employed. A 100-kDa [³H]palmitoylated polypeptide corresponding to the _1b-adrenoreceptor-G₁₁ fusion protein was also observed but only in the positively transfected cells (Fig. 5). When the time course of [³H]palmitoylation of the fusion protein was monitored in the presence and absence of phenylephrine, the rate, but not the maximal extent, of [³H]palmitoylation was markedly enhanced by the agonist (Fig. 6). This effect was specific for agonist, since the presence of phentolamine, an antagonist/inverse agonist at the _1b-adrenoreceptor, did not alter the kinetics or extent of palmitoylation (data not shown). Endogenous G_q/G₁₁ was also [³H]palmitoylated in a time-dependent manner. However, there was no indication that this was regulated significantly by agonist occupation of the fusion protein (Fig. 6), consistent with the notion that the fusion protein does not interact to a significant extent with the endogenous G protein pool. Both the _1b-adrenoreceptor-C9S,C10S G₁₁ fusion protein and the C365A,C367G _1b-adrenoreceptor-G₁₁ fusion protein were also targets for [³H]palmitoylation (Fig. 7, A and B). Each of the three fusion proteins expressed equally well as measured by the specific binding of the high affinity ₁-adrenoreceptor ligand [³H]prazosin (see below). Thus, the lower incorporation of [³H]palmitate into _1b-adrenoreceptor-C9S,C10S G₁₁ and C365A,C367G _1b-adrenoreceptor-G₁₁ (Fig. 7, A and B) reflects the reduced number of sites for acylation in these fusion proteins, which contain the acylation-resistant, mutated G protein and the acylation-resistant receptor, respectively. Furthermore, since the labeling of _1b-adrenoreceptor-C9S,C10S G₁₁ must represent fatty acylation of the receptor, the acylation status of the _1b-adrenoreceptor is clearly regulated by the presence of agonist (Fig. 7, A and B). Equally, palmitoylation of C365A,C367G _1b-adrenoreceptor-G₁₁ must represent labeling of the G protein. This was also regulated by agonist (Fig. 7, A and B) in a concentration-dependent manner with EC₅₀ = 7.8 × 10⁷ M (Fig. 7C).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   The 1b-adrenoreceptor-G11 fusion protein is a target for 3H palmitoylation. HEK293 cells were mock-transfected (1) or transfected to express the 1b-adrenoreceptor-G11 fusion protein (2) and labeled with [3H]palmitate for 2 h. Samples were immunoprecipitated with antiserum CQ, resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   The kinetics and agonist-sensitivity of palmitoylation of the 1b-adrenoreceptor-G11 fusion protein. HEK293 cells were transfected to express the 1b-adrenoreceptor-G11 fusion protein (lanes 1-10) or mock-transfected (lanes 11 and 12) and exposed to [3H]palmitate for 5 (lanes 1 and 2), 15 (lanes 3 and 4), 30 (lanes 5 and 6), 60 (lanes 7 and 8), or 120 min (lanes 9-12) in the absence () or presence (+) of phenylephrine (PE; 10 µM). A, samples were immunoprecipitated and processed as in Fig. 5. B, the autoradiograph to A was scanned, and the intensity of labeling of the 1b-adrenoreceptor-G11 fusion protein with [3H]palmitate was quantitated. The kinetics of labeling in the absence (open symbols) and in the presence (filled symbols) of phenylephrine are shown. Three further experiments produced similar results.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Agonist-regulation of palmitoylation of the 1b-adrenoreceptor and G11. A, the 1b-adrenoreceptor-G11 fusion protein (lanes 1 and 2), the 1b-adrenoreceptor-C9S,C10S G11 fusion protein (lanes 3 and 4), or the C365A,C367G 1b-adrenoreceptor-G11 fusion protein (lanes 5 and 6) were expressed transiently in HEK293 cells. Experiments akin to those of Fig. 6 were then performed in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of phenylephrine with labeling with [3H]palmitate for 30 min. B, incorporation of [3H]palmitate in the samples shown in A was quantitated by densitometry. C, HEK293 cells transiently expressing the C365A,C367G 1b-adrenoreceptor-G11 fusion protein were labeled for 10 min in the presence of varying concentrations of phenylephrine.

To further explore the functionality of the _1b-adrenoreceptor-G₁₁ fusion proteins, the capacity of phenylephrine to stimulate binding of [³⁵S]GTPS was measured. Traditionally, it is difficult to observe significant elevation of [³⁵S]GTPS for receptors that couple to members of the G_q/G₁₁ family (33). This indeed was the case when experiments were performed on membranes expressing _1b-adrenoreceptor-G₁₁, since basal levels of [³⁵S]GTPS binding were high (data not shown). However, by immunoprecipitating the fusion protein from the membranes with antiserum CQ following the [³⁵S]GTPS binding assay, the background was reduced to the extent that a greater than 30-fold stimulation of [³⁵S]GTPS binding by phenylephrine was observed (Fig. 8A). Phenylephrine was without effect in mock-transfected cells (Fig. 8A), and the effect of phenylephrine on [³⁵S]GTPS binding was suppressed completely when experiments were performed in the presence of a high concentration of unlabeled GTPS (Fig. 8B). The _1b-adrenoreceptor-C9S,C10S G₁₁ fusion and the C365A,C367G _1b-adrenoreceptor-G₁₁ fusion were also able to produce effective stimulation of [³⁵S]GTPS binding upon the addition of phenylephrine, and the potency of the agonist (EC₅₀ = 1.2 × 10⁶ M) was unaffected by the palmitoylation potential of either the receptor or the G protein (Fig. 8C). Prazosin is a high affinity antagonist/inverse agonist at the hamster _1b-adrenoreceptor (34). [³H]Prazosin bound to each of the _1b-adrenoreceptor-G₁₁, C365A,C367G _1b-adrenoreceptor-G₁₁, and _1b-adrenoreceptor-C9S,C10S G₁₁ fusion proteins with high affinity (Fig. 9A). Phenylephrine was able to compete with [³H]prazosin for binding to these constructs (Fig. 9B) with similar estimated affinity (K_i of _1b-adrenoreceptor-G₁₁ = 7.2 × 10⁶ M; K_i of C365A,C367G _1b-adrenoreceptor-G₁₁ = 9.1 × 10⁶ M; and K_i of _1b-adrenoreceptor-C9S,C10S G₁₁ = 1.1 × 10⁵ M).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Phenylephrine stimulates the binding of [35S]GTPS to 1b-adrenoreceptor-G11 fusion proteins. HEK293 cells were mock-transfected (A) or transfected to express 1b-adrenoreceptor-wild type G11 (A-C; circles in C), the 1b-adrenoreceptor-C9S,C10S G11 (C; squares), or the C365A,C367G 1b-adrenoreceptor-G11 fusion protein (C; triangles). Membranes were prepared, and the binding of [35S]GTPS was measured in the absence and presence of phenylephrine (10 µM in A and B; varying concentrations in C). Samples were then immunoprecipitated with antiserum CQ and counted. In B, GTPS (100 µM) was added (filled symbols) to suppress binding of [35S]GTPS.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   The affinity of phenylephrine binding to the 1b-adrenoreceptor is unaffected by the palmitoylation potential of G11. A, the specific binding of a range of concentrations of [3H]prazosin to the 1b-adrenoreceptor-wild type G11 (circles), the 1b-adrenoreceptor-C9S,C10S G11 (squares), or the C365A,C367G 1b-adrenoreceptor-G11 (triangles) fusion proteins expressed transiently in HEK293 cells was measured. Analysis of such saturation isotherms demonstrated that [3H]prazosin bound with similar affinity to each. B, the binding of [3H]prazosin (0.5 nM) to the 1b-adrenoreceptor-wild type G11, the 1b-adrenoreceptor-C9S,C10S G11, or the C365A,C367G 1b-adrenoreceptor-G11 fusion proteins was competed for by varying concentrations of phenylephrine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Both GPCRs and G protein  subunits are targets for post-translational palmitoylation (14-16). This can be a regulated process and agonist occupation of a GPCR may enhance the rate of turnover of [³H]palmitate labeling in both the GPCR and the G protein activated by the GPCR (35, 36). There are, however, a variety of technical limitations inherent in studies that attempt to examine GPCR-mediated regulation of G protein palmitoylation. Significant among them are lability of the thioester link between the fatty acid and the protein, the length of time usually required to produce autoradiograms of appropriate intensity when using [³H]palmitate as the label and the possibility that observed effects predominantly reflect alterations in the specific activity of the palmitoyl CoA pool (37, 38). A further issue is that G proteins are often present in marked excess compared with a GPCR in cells (39, 40). Thus, if only a small fraction of the total pool of a G protein is activated by a GPCR, it may be difficult to detect agonist-mediated regulation of palmitoylation of this fraction. One method to overcome the latter issue is to ensure that only G proteins activated by the GPCR are isolated and analyzed.

To do this for the pairing of the _1b-adrenoreceptor and G₁₁, a fusion protein was constructed in which the N terminus of G₁₁ was linked to the C-terminal tail of the GPCR such that a single open reading frame was generated containing the sequences and functions of both partner proteins. Following expression of this fusion protein and labeling of cells with [³H]palmitate in the presence or absence of the ₁-adrenoreceptor agonist phenylephrine, the fusion protein could be effectively immunoprecipitated and analyzed using an antiserum that identifies the C terminus of G₁₁. Since the receptor-G protein fusion protein has a predicted molecular mass of some 100 kDa, it is extremely well resolved following SDS-polyacrylamide gel electrophoresis from the endogenous G_q/G₁₁ expressed by most cells. This approach allowed clear demonstration that agonist enhanced the kinetics of turnover of [³H]palmitate of the fusion protein but not the maximal extent of labeling (Fig. 6). Although such results are consistent with reports on agonist effects on co-expressed ₂-adrenoreceptor and G_s (17-19), they are very different from those reported for a ₂-adrenoreceptor-G_s fusion protein (41), in which the palmitoylation of the fusion protein was greatly reduced in the presence of agonist ligands. The basis for this variation is unclear, particularly since Loisel et al. (41) have suggested that repalmitoylation of the depalmitoylated ₂-adrenoreceptor-G_s fusion protein is inhibited and that this might be related to the poor capacity of this construct to become desensitized. Interestingly, however, we have observed that agonist also greatly accelerates the kinetics of acylation of a 5-hydroxytryptamine 1A-receptor-G_o1 fusion protein,² consistent with observations of an enhanced rate of [³H]palmitoylation of G_i when activated by a co-expressed 5-hydroxytryptamine 1A receptor (42).

When the _1b-adrenoreceptor-G₁₁ fusion protein was expressed in HEK293 cells, which express both G₁₁ and G_q endogenously, these G proteins also became [³H]palmitoylated in a time-dependent manner. However, this was not modified by agonist treatment, indicative that the endogenous G proteins were not accessed by the fusion protein to a significant extent. Further confirmation of functional activation of the fused G₁₁ by the receptor was obtained in [³⁵S]GTPS binding studies. Such assays provide a direct monitor of receptor-mediated guanine nucleotide exchange on a G protein. It is traditionally difficult, however, to monitor such an effect of agonists for G_q family G proteins because of the high background contributed by other G proteins (33). This was also the case in the current studies when such assays were performed on membrane preparations. However, immunoprecipitation of the fusion protein allowed stimulation to be observed easily. Although the low basal guanine nucleotide exchange rate of the G_q family G proteins is often cited as a limitation in efforts to monitor agonist stimulation of [³⁵S]GTPS binding to them, in the immunoprecipitation approach employed herein, it is a distinct advantage, since there was very little radioactivity bound to the fusion protein in the absence of agonist. When equivalent studies were performed in EF88 cells transfected with the _1b-adrenoreceptor-G₁₁ fusion protein, immunoprecipitation also provided direct evidence that the agonist stimulation of [³⁵S]GTPS binding was on the G₁₁ of the fusion protein (data not shown).

Phenylephrine stimulation of [³⁵S]GTPS binding was achieved with an EC₅₀ of 1.2 × 10⁶ M (Fig. 8). This is some 6-fold more potent than the estimated K_i for phenylephrine for the _1b-adrenoreceptor-G₁₁ fusion protein calculated from the ability of this agonist to compete with [³H]prazosin for binding to the fusion protein (Fig. 9). Since the 1:1 stoichiometry of the protein partners of the fusion predicts that activation of the G protein would require occupancy of the receptor by agonist, the basis for this difference is currently uncertain. This may suggest that the GPCR is not restricted to stimulating only the G protein that is directly linked to the receptor, but further analysis will be required to explore this possibility. However, it should also be borne in mind that the conditions required for the two assays are not identical, and although we tried to perform the binding assays in buffer conditions similar to those of the [³⁵S]GTPS binding assay, subtle variations may alter estimates of ligand affinity and potency in different assays. It was noticeable, however, that in both assays elimination of the acylation sites in either the receptor or G protein did not alter these parameters for phenylephrine (Figs. 8 and 9).

Direct evidence of the capacity of the wild type fusion protein to regulate downstream end points was obtained following its expression in EF88 cells. Since these cells are derived from a mouse embryo in which the genes for both G₁₁ and G_q were inactivated, signal cannot proceed via endogenous forms of these G proteins. The addition of phenylephrine resulted in an elevation of intracellular [Ca²⁺]. This was derived from intracellular Ca²⁺ stores, since all such assays were performed in the absence of extracellular Ca²⁺. Co-expression of transducin  with the _1b-adrenoreceptor-G₁₁ fusion protein fully blocked the phenylephrine-induced rise in [Ca²⁺]. Since transducin  is used routinely as a · complex-sequestering agent, these results demonstrate that signal was transduced via ·. Such results prove that the _1b-adrenoreceptor-G₁₁ fusion protein can both interact with and release · in response to agonist stimulation. Furthermore, since the co-expression of transducin  also fully attenuated signal from the _1b-adrenoreceptor-C9S,C10S G₁₁ fusion protein, the lack of palmitoylation of the G protein  subunit prevents neither binding nor agonist-mediated release of · complex. Although previous studies have indicated that a fusion protein between the ₂-adrenoreceptor and G_s can co-immunoprecipitate · complex (43), this is the first study that has convincingly shown agonist-mediated signaling produced by · derived from a GPCR-G protein fusion protein. In the single cell imaging studies, elevation of [Ca²⁺] appeared to occur somewhat more rapidly for the fusion protein incorporating the wild type G protein compared with the one containing the palmitoylation-resistant form of G₁₁ (Fig. 3). EF88 cells are difficult to transfect, and thus we used single cell Ca²⁺ imaging in concert with co-expression of green fluorescent protein for these sets of studies because only a small fraction of the cells in any particular field were positively transfected. Thus, although we noted that overall levels of expression of the two forms of the fusion protein, monitored by binding of [³H]prazosin, were similar following transfection of HEK293 cells for the [³H]palmitoylation and [³⁵S]GTPS binding studies, we do not know the levels of expression of the constructs in the individual EF88 cells that were imaged. Since each copy of the construct can release one copy of the · complex, higher levels of the fusion proteins in a particular cell might be expected to result in more rapid kinetics of liberation of [Ca²⁺]_i. Overall, these time courses were not very different. It is also noteworthy that Ca²⁺ elevation in cells expressing any of the forms of the fusion protein was significantly slower than following co-expression of the isolated receptor and G protein and, more importantly, that the kinetics of Ca²⁺ elevation were considerably slower when the _1b-adrenoreceptor was co-expressed with either C9S G₁₁ or C10S G₁₁ compared with the wild type G protein (Fig. 2). Both of these mutants have been shown to partition between membrane and cytosol much less effectively than wild type G₁₁ (20, 21), and this thus restricts their relative membrane concentration compared with the receptor.

It is also of considerable interest that interactions with · are thought to be required for membrane targeting and palmitoylation of both G_q and G_s (26) and play key roles in the targeting of G subunits to the correct membrane compartments with lipid acylation and then stabilizing membrane association (44, 45). In the native state, there is a complex interplay between the palmitoylation of G protein  subunits and interaction with the · complex. For example, mutants of G_q unable to bind · are largely soluble and as such are not palmitoylated (26), since this acylation appears to occur at the membrane. Tethering of the  subunit to the membrane is potentially sufficient to allow palmitoylation, however, since the addition of an N-terminal myristoylation signal to the / interaction-defective mutants of G_q allowed their palmitoylation but did not restore / interaction (26). These two processes thus can clearly be resolved. Detailed review of this topic has recently been provided (46). In the current studies, we have used attachment to a receptor to direct trafficking of the palmitoylation-resistant form of G₁₁ to the membrane and to demonstrate that / interaction does not require prior palmitoylation. This is consistent with the concept that / interaction can stabilize the localization of a nonacylated G protein at the membrane until it becomes palmitoylated, which then provides extra anchorage, and with older studies, which indicated that high level overexpression of · complex can assist with trafficking of acylation-resistant forms of G to the membrane (see Ref. 46 for details). Previous studies on G_i1 and G_z, which like the other G_i family G proteins are modified by both palmitoylation and myristoylation at their N terminus, have also shown that lack of palmitoylation does not impair GPCR-mediated release of · (47, 48). However, palmitoylation of G_s has been reported to increase its affinity for · by some 5-fold (49). Others have used different strategies to tether a G protein at the membrane. These have included linking the G protein to a single transmembrane-spanning element of a GPCR (50). However, the current approach ensures proximity of the G protein to the GPCR and has been successfully applied previously for acylation-resistant forms of G_i1 (51).

The capacity of phenylephrine to stimulate the binding of [³⁵S]GTPS binding to fusion proteins in which the acylation sites in either the receptor or G protein were mutated indicates that Cys residues at positions 9 and 10, and thus potential palmitoylation, is not inherently required for information transfer from the _1b-adrenoreceptor to the G protein. Furthermore, since the binding affinity of phenylephrine was little affected by these mutations (Fig. 9), it is not surprising that the measured EC₅₀ for the agonist to stimulate [³⁵S]GTPS binding was not different between the various fusion proteins employed (Fig. 8C).

The vast majority of rhodopsin-like GPCRs have one or more Cys residues in the first 20 amino acids of the predicted C-terminal tail. In many cases, these have been shown directly to be sites for palmitoylation (36, 52-55). Mutation of these residues has been reported to have effects ranging from alterations in G protein coupling (52-54) and receptor internalization/membrane delivery (55, 56) to effects on the phosphorylation of the GPCR by regulatory kinases (36, 57) and interactions with -arrestin-1 (58). The _1b-adrenoreceptor-C9S,C10S G₁₁ fusion protein was also able to incorporate [³H]palmitate. This therefore must represent acylation at Cys³⁶⁵ and/or Cys³⁶⁷ of the receptor. The kinetics of incorporation of [³H]palmitate into this form of the fusion protein was also enhanced by treatment with phenylephrine, indicating that turnover of receptor palmitoylation is not a process that has to be integrated with equivalent regulation of the acylation status of the G protein (Fig. 7). Equally, agonist regulated palmitoylation of the C365A,C367G _1b-adrenoreceptor-G₁₁ fusion protein and thus of the G protein activated by the receptor.

The current studies provide new insights into the coordinated agonist regulation of post-translational acylation of a GPCR and its associated G protein. Furthermore, they introduce a strategy to monitor the true extent of receptor-enhanced [³⁵S]GTPS binding to G_q family G proteins. Finally, they also provide novel insights into the mechanism of [Ca²⁺] signaling in cells lacking the  subunits of the G proteins which are usually assumed to transduce these signals in cells.

	ACKNOWLEDGEMENT

EF88 cells were a kind gift from Dr. M. I. Simon.

	FOOTNOTES

^* Financial support for this work was provided by the Wellcome Trust and Medical Research Council (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Scottish Biomedical, Todd Campus, West of Scotland Science Park, Glasgow G20 OXA, Scotland, United Kingdom.

^§ To whom correspondence should be addressed: Davidson Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom. Tel.: 44 141 330 5557; Fax: 44 141 330 4620; E-mail: g.milligan@bio.gla.ac.uk.

Published, JBC Papers in Press, July 18, 2001, DOI 10.1074/jbc.M103816200

² P. A. Stevens, P. Welsby, E. Kellett, and G. Milligan, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; GTPS, guanosine 5'-3-O-(thio)triphosphate; PCR, polymerase chain reaction.

	REFERENCES

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